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VS Memorandum (VSM) 800.211 

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Guidance for Master Reference Qualification, Requalification, Dating, and Monitoring

1. Purpose and Background 

Many relative potency assays used for serial release testing of veterinary biologics rely on 

the use of a reference preparation that was qualified, directly or indirectly, in a hostanimal vaccination/challenge study. Title 9, Code of Federal Regulations (9 CFR), section 

113.8 (d) indicates that the lot of reference used to determine relative antigenic content 

must have an initial dating period equal to the dating of the product or as supported by 

data acceptable to the Animal and Plant Health Inspection Service (APHIS). The dating 

period of the Master Reference (MR) and Working Reference (WR) may be extended if 

data are provided to support that the minimum potency of the Master Reference is 

adequately above the minimum level needed to provide protection in the host animal.

In 2011, the Center for Veterinary Biologics (CVB) published VSM 800.211, Guidance for 

Master Reference Qualification and Requalification, to revise the policy for determining 

the dating period for Master References of certain products, historically referred to as 

“previously licensed products”, to reduce required animal testing and lessen the burden 

of time consuming and resource intensive vaccination/challenge studies. The intention 

was to limit the time and resources devoted to maintaining assays for licensed products

and encourage the development and validation of assays for products licensed 

thereafter. VSM 800.112, Guidelines for Validation of In Vitro Potency Assays, was also

updated in 2011 to include Appendix III, Guidance for Validating ELISA Relative Potency 

Assays, which includes guidance on monitoring and Master Reference dating for products 

licensed after January 1, 2011. 

This memorandum describes the expectations for monitoring the stability of MRs for 

legacy and modern products. This purpose of this document is to preserve the guidance

in the 2011 version of VSM 800.211 for what were called “previously licensed products” 

and additionally, to update and clarify the guidance in VSM 800.112 Appendix III and VSM 

800.211 pertinent to dating and stability monitoring of MRs of products licensed since 

January 1, 2011. This updated memorandum also includes guidance on reference 

qualification and requalification. This document also includes information from VSM 

800.90, Guidelines for Veterinary Biological Relative Potency Assay and Reference 

Preparations Based on ELISA Antigen Quantification, which was removed from the CVB 

website in 2016. 

2. Document Status 

A. Issue Date: 2/8/2023 

B. This document replaces VSM 800.211 dated June 28, 2011, which is cancelled. 
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3. Reason for Reissuance 

This memorandum has been updated to incorporate contemporary information 

regarding dating and reference stability monitoring for all references. 

4. Authority and References 

A. Authorities

7 CFR 371.4

9 CFR 113.8 

B. References

• VSM 800.112, Guidelines for Validation of In Vitro Potency Assays 

• VSM 800.206, General Licensing Considerations: Preparing Outlines of Production 

for Vaccines, Bacterins, Antigens, and Toxoids and Diagnostic Test Kits 

 

C. Definitions

1) 9 CFR 101.5 (o) Master Reference: A reference whose potency is correlated, 

directly or indirectly, to host animal immunogenicity. The MR may be used as the

working reference in in vitro tests for relative potency. The MR may also be used 

to establish the relative potency of a serial of product used in requalification 

studies and to establish the relative potency of working references. The 

preparation of an MR as described in a filed Outline of Production (OP) may be: 

• A completed serial of vaccine or bacterin prepared in accordance with a filed 

OP; 

• A purified preparation of a protective immunogen or antigen; or 

• A nonadjuvanted harvested culture of microorganisms. 

2) 9 CFR 101.5 (p) Working Reference: The reference preparation that is used in 

the in vitro test for the release of serials of product. Working References 

(WRs) may be: 

• The Master Reference; or 

• A serial of product that has been prepared and qualified, in a manner 

acceptable to APHIS for use as a reference preparation. 

WRs used in relative potency assay methods must have a relative potency 

≥ 1.0 when compared to the MR. The geometric mean potency of the WR when 

compared to the MR must be established in the approved potency assay by five 

or more independent assays.
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3) 9 CFR 101.5 (q) Qualifying Serial (QS): A serial of biological product used to 

test for immunogenicity when the Master or Working Reference is a purified 

antigen or nonadjuvanted harvest material. Qualifying serials must: 

• Be produced in accordance with the filed OP. 

• Be tested for immunogenicity. 

• Have a geometric mean relative potency, when compared to the Master 

Reference, of not greater than 1.0 as established by: 

o Independent parallel line assays with five or more replicates; or 

o Other valid assay methods for determining relative antigen content which 

demonstrate linearity, specificity, and reproducibility at least equivalent to 

the parallel line assay and are acceptable to APHIS. 

• Be within its permitted dating period. 

4) 9 CFR 101.5 (r) Immunogenicity: The ability of a biological product to elicit an 

immune response in animals as determined by test methods or procedures 

APHIS finds acceptable. 

In this document immunogenicity typically refers to the demonstration of 

efficacy by means of a host animal vaccination/challenge study.

5) Internal control (IC): A preparation included in an assay to serve as an 

independent measure of the assay’s performance, but which does not serve 

as an assay validity control. Internal controls may be crude preparations, semipurified or purified fractions containing the target analyte, or other materials 

that respond similarly to the reference in the assay. ICs may be used as 

surrogates to monitor the stability of a reference. 

6) Serial release assay: The test method used for determining the potency of a 

test serial as described in Section V. C. of the Outline of Production. 

7) Relative potency assay (RPA): A serial release assay for which the potency of a 

test serial is compared to that of a reference preparation. 

8) Reference stability monitoring (RSM) assay: A test method used to monitor 

the stability of some aspect of the reference, qualitative and/or quantitative. 

RSM assays are used to monitor the quantitative and qualitative aspects of a 

reference preparation, not a serial of product. RSM assays are different than 

the serial release assay. 

 
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9) Major manufacturing change: A change to the production of a biological 

product administered to animals that would require a new efficacy and/or 

field safety study prior to approval of the change. 

10) Analytical principle: The physical/biological/chemical/biochemical approach 

for measuring a target of interest and the metric used. Examples include:

• A serological test measuring an antibody response to vaccination and the 

titer used to express the amount of antibody present; or 

• A sandwich ELISA measuring the potency of OspA relative to a Borrelia 

burgdorferi reference. 

11) Legacy Products: A category of veterinary biological products (not to include 

diagnostic test kits) licensed or permitted before January 1, 2011, provided 

that no major changes in the manufacturing process or serial release assay

have been implemented. Historically these were termed “Previously licensed 

products.” 

12) Modern Products: This is a category of veterinary biological products (not to 

include diagnostic test kits) licensed or permitted on or after January 1, 2011. 

This category also includes legacy products that have been reclassified due to

a major manufacturing change as defined above, or a change to the analytical 

principle of the relative potency assay (RPA) that occurred on or after January 

1, 2011. These products were previously termed “Newly licensed products.” 

13) Time Zero: Time zero refers to the date animals are first vaccinated in a study 

to qualify a MR (i.e., demonstrate the efficacy of a proposed reference). 

14) Forced degradation by freeze-thaw study (FDFT): A study using a specified 

freeze-thaw regimen (described in Section 6. I. below) to determine if the MR

has degraded resulting from stress induced by repeated freezing and thawing. 

5. Audience 

VS employees and members of the biologics industry.

 
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6. Guidance 

The following sections provide specific details regarding dating, monitoring, and 

qualification/requalification of MRs and WRs.

A. Identifying the Master and Working References

Identify both the MR and the WR including storage conditions in the filed OP. See 

VSM 800.206 for detailed guidance. 

B. Storage Conditions of MRs

Store the MR under constant defined conditions. A Standard Operating Procedure 

(SOP) must describe a system for recording the storage conditions and verifying that 

they do not deviate from the specific limits defined in the SOP. Use calibrated 

equipment to record the temperature at regular intervals by automated or manual 

means. Store data electronically and make it available for inspection. Promptly notify 

CVB of any deviation in the allowable range of storage conditions. 

C. Master Reference Qualification and Requalification via Host Animal Immunogenicity 

Study 

Qualification of a MR for use in an in vitro relative RPA is either the initial 

establishment of a MR or the establishment of a new MR to replace an existing MR. 

The MR may be directly qualified in animals when it is a non-frozen product 

reference. If the MR is 1) a frozen product reference, 2) a stabilized and frozen 

preparation, or 3) a purified preparation, a QS will be needed for MR qualification or 

requalification.

When a QS must be used, test the MR and the QS in a minimum of five independent 

replicate serial release assays to establish the RP of the QS or to determine the 

dilution of the MR that would result in the QS having an RP of 1.0.

Prior to its expiration or any time there is an indication of lack of stability, a MR must 

be requalified, or a new MR must be qualified by conducting a host animal 

immunogenicity study unless qualification in vitro is allowed (Section 6. D.). The same 

animal model and efficacy study design that supported initial licensure or the most 

recent reference qualification may be used. A similar outcome to either study will be 

sufficient to qualify or requalify an MR. If the licensee or permittee elects to propose 

improvements to the study design used previously, the protocol should explain and 

justify the proposed changes. A new FDFT study (Section 6. I.) must be conducted for 

a modern product frozen MR every time a MR is qualified or requalified.

 
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D. Master Reference Qualification In Vitro

If a MR is monitored from time zero for stability using validated RSM assays in an 

approved RSM plan, a new MR may be qualified in vitro using the RSM assays upon 

inventory depletion or degradation of the existing MR. Most MRs qualified prior to 

the date of this memorandum will require one additional host animal immunogenicity

study to qualify a MR that is tested via an approved RSM plan from time zero. An 

allowance for in vitro qualification will be made if a newly approved monitoring plan 

is implemented and time zero testing is conducted within 2 years of the host animal 

immunogenicity study used to qualify the MR.

To be eligible for in vitro qualification of a MR, establish a baseline at time zero. Test 

20 vials of the MR at time zero in the quantitative RSM assay to establish the 

Baseline 1 discussed in Section 6. H. Estimate the mean and 90 percent confidence 

interval from testing the 20 vials in the quantitative RSM assay. The lower bound of 

this 90 percent confidence interval sets the baseline for in vitro qualification in the 

future (Baseline 1 in Section 6. H.). CVB will consider proposals for establishing the 

baseline for in vitro qualification using alternative methods. This baseline is treated as 

a non-inferiority margin for the statistical analysis when qualifying a new MR using 

the in vitro testing approach. To qualify a new MR using the quantitative RSM assay, 

test a sufficient number of vials and estimate the mean and 90 percent confidence 

interval. If the lower bound of this confidence interval remains above the margin 

(baseline) established at time zero, the new MR will be qualified. The precision 

analysis from the RSM assay validation can be used to help determine a sufficient 

number of vials to test (Section 6. J.).

Test the MR using the qualitative assay to demonstrate no unexpected results.

In vitro MR qualification will not be an option when the MR stability is monitored 

using an IC in the serial release assay. RSM assays for the IC are required to ensure 

the IC is not degrading at the same rate as the MR, which would not necessarily be 

detected in the serial release assay. The MR is not tested using RSM assays and 

therefore cannot be qualified using an in vitro approach.

E. Working Reference Qualification

When a separate WR is prepared, demonstrate parallelism between the MR and WR 

in the serial release assay.1 Establish the geometric mean potency of the WR when 

compared to the MR in the approved serial release assay by five or more 

independent test runs. The WR must have a geometric mean RP no less than 1.0 

when compared to the approved MR.

1 Demonstrating similarity between the MR and WR may be necessary for other types of assay systems.
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F. Dating & Associated Monitoring Requirements for Master References

1) Dating for Legacy Product MR. MRs for legacy products may be used continuously 

for serial release (i.e., as a WR) or to establish new WRs for up to 15 years from 

the date of initial qualification (or requalification) via host animal immunogenicity

study if there are no obvious changes in the behavior of the MR in the serial 

release assay (Section 6. G.). MRs exhibiting an obvious change in behavior in the 

serial release assay at any time must be replaced. No dating beyond 15 years will 

be considered under this scenario unless the MR is requalified in a host animal 

immunogenicity study (Section 6. C.).

Licensees and permittees may elect but are not required to use procedures 

described in Section 6. H. to monitor MRs for legacy products, which would allow

longer dating periods for MRs of legacy products without new host animal

immunogenicity studies and allows for the possibility of in vitro qualification.

2) Dating for Modern Product Frozen MR. Evaluate all modern product frozen MRs in 

a FDFT study (Section 6. I.). The results of this study will determine if, in addition 

to serial release trending data, monitoring the MR by RSM assays is necessary.

a. MRs of modern products that are stored frozen and pass the FDFT study can

be used for a period of 15 years. If the licensee or permittee chooses to 

monitor using only serial release trending data, then the MR would not be 

eligible for dating beyond the 15 years without requalifying the MR or 

qualifying a new MR in a host animal immunogenicity study. If the licensee or 

permittee chooses to monitor using RSM assays, they should follow the same 

time frames provided in Section 6. F. 2) b. If time zero testing using the RSM 

assays is conducted within 2 years of qualification, the MR will be eligible for 

dating beyond 15 years with the possibility of in vitro qualification.

b. MRs of modern products that are stored frozen and do not pass the FDFT 

study will initially be granted 5 years dating. The dating can be increased in 5-

year increments to a total of 15 years, contingent upon submission of serial 

release trending data at 2 1⁄2 year intervals and the milestones described 

below. Prior to the final expiration, qualify a new MR or requalify the current 

MR in a host animal immunogenicity study (Section 6. C.). This step is 

necessary so that the qualified/requalified MRs can be tested according to the 

approved RSM plan with validated RSM assays from time zero.

1. Submit a conceptual RSM plan for review within the first 2 1⁄2 years

(Section 6. H.). Revise as necessary based on feedback from CVB.
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2. The RSM plan must be tentatively approved within the first 5 years. Upon 

tentative approval, the MR expiration will be extended for an additional 5 

years (for a total of 10 years from time zero). Final approval is subject to 

successful validation of the RSM assays.

3. Validate the RSM assays and obtain CVB approval before the end of the 

current (10-year) expiration. Upon approval of the RSM assays, the 

expiration of the MR will be extended 5 years (for a total of 15 years). 

3) Dating for Modern Product Non-Frozen MR. MRs of modern products that are not 

stored frozen will be given product dating prior to the approval of the RSM plan 

and validation of the RSM assays. There is no option to extend dating beyond 

product dating based on the development of the RSM plan and assay validation 

efforts. Once a non-frozen MR can be tested from time zero using validated RSM 

assays, such MRs may be eligible for indefinite dating with demonstration of 

stability.

4) Dating for WRs. If a WR is serial of product, stored according to the label, it will 

receive product dating. See Section 6. E. for details on qualifying new WRs.

G. Monitoring Master Reference Stability Using Serial Release Trending Data

The only monitoring requirement for any legacy MR to be eligible for 15-year dating is

using serial release testing data from the time of initial qualification. The monitoring 

of any modern MR should include serial release testing as well, which may be all that 

is required for monitoring (Section 6. F. 2. a.) or may be just one component of the 

monitoring (Section 6. F. 2. b.). 

Reports summarizing all serial release data from time zero must be submitted to CVB

Policy, Evaluation and Licensing (CVB-PEL) at 2 1⁄2 year intervals. A submission should 

be made to CVB-PEL including all raw data from the MR, the WR (if different than the 

MR), test serials, and controls in an electronic format consistent with the CVB Data 

Guide, available on CVB’s website. Include all data from the initial reference 

qualification through the most recent serial release test. The report should include 

graphical and/or tabular summaries of the data as appropriate and a summary 

statement of the storage conditions and temperature monitoring procedure for the 

MR (and WR if different than the MR). Test the MR a minimum of once every quarter

in the serial release assay. This will be necessary when the WR is different than the 

MR or if no serials are tested for serial release during such time.
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The raw data sets should be complete. For example, ELISA raw data would include the 

optical density for every well on each plate, along with the related information, such 

as plate layout and dilution sequences. For serological assays, include serum titers of 

each animal and for laboratory animal challenge tests, include the response of every 

animal, including those used for back titration of the challenge material. 

Where appropriate, data summaries should include plots of each individual test. This 

is important, for example, with test methods using dilution sequences, such as ELISAs 

or animal vaccination-challenge tests. The plot should include the reference and 

serials. In many situations, such as ELISAs, it may also be useful to plot the parameter 

estimates from regression models fit to individual preparations on each test, as well 

as the ratio of parameter estimates comparing those from the serials to those from 

the reference. 

H. Master Reference Monitoring Using Reference Stability Monitoring Assays

To monitor MRs via RSM assays, whether required or optional, the licensee or 

permittee must establish an acceptable RSM plan with validated RSM assays. The 

licensee or permittee is encouraged to submit a plan before submitting the assays. 

The plan should include details of the proposed assays, frequency of testing, MR 

storage conditions, and method of monitoring the storage conditions. There is no 

expectation that the assays will be validated at the time the plan is submitted, but 

CVB review at this time will allow preliminary feedback and preliminary acceptance if 

the plan appears reasonable. 

The assays in a RSM plan must monitor the quantitative and qualitative 

characteristics of the MR. Some assay methods might be capable of fulfilling both 

objectives, but multiple assay methods may be necessary. See Section 6. L. for 

additional considerations for RSM assays. All RSM assays must be validated prior to 

final CVB approval and implementation of the RSM plan.

In addition to typical validation parameters (see VSM 800.112), RSM assays must be 

evaluated for their ability to detect degradation of the specific MR they are intended 

to monitor. For a product in which potency is measured by a RPA, the assumption is 

that any preparation with the same or more antigen (protective antigen targeted in 

the assay) will be efficacious in the host animal when prepared in a manner 

consistent with the QS. At the time the pivotal efficacy study is conducted (time zero), 

the amount of antigen considered to be efficacious is established, and a benchmark is 

established regarding the “signal” produced in the RPA. The goal of using RSM assays 

is to assess whether the benchmark has changed.2 Specifically, the objective is to 

2 It is not possible to observe the real quantity of interest, namely the reference at any point in time compared to 

itself at time zero tested in the same run of the assay.
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assure that the signal of the reference in the RPA does not change over time, thus 

assuring the potency of a serial is consistently measured. If the signal does change, it 

is assumed the reference is changing. Therefore, two key baselines must be 

established: 

• Baseline 1: Performance specifications for the MR at time zero which will serve as 

a non-inferiority margin for future in vitro qualification. 

• Baseline 2: Performance specifications when the MR has degraded to the extent 

that it is no longer acceptable for use in serial release testing. 

Additional guidance regarding these benchmarks is covered in Sections 6. D. and 6. J, 

respectively. Once CVB accepts the validation reports for the RSM assays described in 

the RSM plan, you may implement the RSM plan. Submit reports to CVB every 2 1⁄2 

years along with the serial release trending data.

I. Forced Degradation by Freeze-Thaw

At the time of qualification of a modern product frozen MR perform an FDFT study 

using a validated serial release assay. Evaluate the data for a change in the shape of 

the curve and a decrease in the relative potency. This is only necessary for MRs 

qualified in host animal immunogenicity studies.

Subject 10 vials of the MR to the following freeze-thaw regimen (henceforth referred 

to as “treated reference”):

• Vial volumes should represent how the reference is stored, ideally in small 

aliquots (e.g., 1 mL).

• Freeze at normal storage conditions (e.g., -70 to -80 °C) for at least 6 hours to 

ensure complete freezing. 

• Thaw at 20-25 °C until the vial is fully thawed or at least 60 minutes, whichever is 

longer.

• Complete 10 cycles of freezing and thawing as specified above.

• After the final freezing cycle, conduct 10 independent serial release potency tests, 

where one vial of treated reference and one vial of untreated reference are 

tested in each run of the assay.

Evaluate the data for a change in the shape of the dose-response curve and a 

decrease in the relative potency. For relative potency ELISA assays, assess parallelism 

in a manner consistent to assay validation (see VSM 800.112 Appendix III Section 

2.2.5 and Parallelism Analysis in Assay Validation (STATWI0006)). If the treated 

reference passes the parallelism assessment, evaluate the change in potency and 

compare the lower limit of a 90 percent confidence interval estimate of the relative 

potency of the treated reference to a value of 0.8. An MR is determined to pass the 

FDFT study if the treated reference passes the parallelism assessment and the lower 
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limit of the confidence interval for the RP remains above 0.8. MRs that do not do not 

pass the FDFT study are dated according to Section 6. F. 2. b.

J. Ability of a Reference Stability Monitoring Assay to Detect MR Degradation

A critical step in validating assays for an RSM plan is demonstrating the assay’s ability 

to detect degradation in the MR prior to unacceptable signal loss in the serial release 

test. This requires testing MRs at various degrees or levels of degradation during RSM 

assay validation.

Licensees or permittees may “age” an MR artificially by means other than time. If 

repeated freezing and thawing (Section 6. I.) can degrade the reference (which may 

require more than 10 cycles), use this method as it is the most likely type of stressor 

to which a frozen reference will be exposed. If not, harsher methods such as heat or 

chemical treatment may be proposed. Diluting the reference is not an acceptable 

surrogate for degradation. Describe in the RSM assay validation protocol how the 

selected method of forced degradation will mimic natural degradation over time 

under the proposed storage conditions. Vials will be tested after different levels of 

degradation, such as different numbers of freeze and thaw cycles. 

Test a panel of MR vials degraded to various degrees in each proposed RSM assay and 

in the serial release assay. MR degradation may manifest itself in various ways:

Quantitative, qualitative, or both. 

The simplest scenario is where the detected change is quantitative, and the 

relationship between the response in the quantitative RSM assay and the response 

from the serial release assay is linear. When this is the case, establish a 

threshold/baseline (on the fitted line) corresponding to the response of the degraded 

MR that produces an expected RP of 0.8 in the serial release assay (baseline 2 from 

Section 6. H.). If degradation manifests in other ways or the relationship between the 

response from the quantitative RSM assay and the serial release assay is not linear, 

alternative metrics are necessary (such as parameter ratios) and other cutoff test

values may be used. Licensee or permittee must take responsive action, such as 

qualifying a new MR prior to the MR producing a value below the threshold/baseline

in the RSM assay.

When establishing the dose-response relationship, evaluate precision by testing 

multiple vials of the degraded MR in both the serial release assay and the RSM assays. 

The precision of the RSM assay should be considered when determining the number 

of vials of the MR to test at future time points.
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K. Using an Internal Control to Monitor Reference Stability

There may be instances in which an IC is used as a surrogate to monitor stability of 

the MR. In these instances, the IC is tested with the RSM assays and the relationship 

between the IC and the MR in the serial release assay is monitored. Two baselines are 

necessary in this instance, one regarding the IC’s response in the RSM assays and one 

related to the relationship between the IC and the MR in the serial release assay.

To establish the baseline in the RSM assays, test the degraded IC (at various levels) in 

the RSM assays and against the MR in the serial release assay. Establish the 

relationship between the response in the RSM assays and the metrics used to 

monitor the MR in the serial release assay. Establish a baseline in a similar manner to 

the description provided in Section 6. J.

The second baseline will be pre-determined and related to the response in the serial 

release assay (relationship between the IC and the MR). For an ELISA, that baseline 

may be related to a ratio (such as upper asymptote or scale parameters) or to a 

difference in location parameter estimates. Propose a baseline in a protocol and 

justify its value. Conduct a study to demonstrate the baseline value may be generated

(or spanned) using various degrees of a degraded MR.

L. Other Considerations when selecting the RSM assays

1) The monitoring assays must be able to detect a signal change in the serial release 

assay, whether that manifests only as a loss in relative potency or in other ways, 

such as a change in the shape of the dose-response curve.

2) Ideally, but not necessarily, the RSM assays will incorporate the antibody used in 

the serial release assay. This will allow an assessment of the antigen-antibody 

interaction. A measurement of total protein will not be considered as a 

quantitative metric for RSM.

3) If the RSM assays rely on the use of another preparation, such as a standard, 

consider the source of the standard and the frequency and method of 

replacement. Standards that are commercially available with a certificate of 

analysis and defined expirations will be more appropriate than an in-house 

created preparation that could be degrading at a similar rate as the MR.

M. Legacy Status for Combination Products and Major Manufacturing Changes

Major changes in the manufacturing procedure will result in a product licensed prior 

to January 1, 2011, being classified as a modern product for the purposes of this 

guidance. An example would be changes that require a new product code and a new 

efficacy and/or safety study before approval of the change.
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Combination products containing a legacy product and a modern product will be 

treated as a modern product for the new antigens, and as a legacy product for those 

antigens licensed prior to January 1, 2011, if the combination of fractions does not 

impact the potency tests for the legacy licensed fractions. Multivalent products 

combining antigens of legacy products will be evaluated on a case-by-case basis.

Improvements to the serial release potency assays of legacy products will not cause a 

legacy product to be reclassified if such changes do not alter the analytical principle 

of the test method. Modifications that improve the performance of a legacy product’s 

test method will not require complete revalidation of the assay.

N. Summary of Dating, Monitoring, Reference Qualification

This section is intended to provide highlights on legacy and modern product MRs

regarding monitoring methods, dating, and reference qualification. CVB intends the 

information here to help simplify and clarify guidance; it does not intend it to be all 

inclusive.

1) Scenario 1

• Legacy product MRs

• Monitoring via serial release data only, submitted every 2 1⁄2 years

• 15-year dating maximum

• Host animal immunogenicity study required to qualify a new MR or requalify 

the current MR

2) Scenario 2

• Modern product MRs stored frozen

• Passes FDFT regimen

• Monitoring via serial release data only, submitted every 2 1⁄2 years

• 15-year dating maximum

• Host animal immunogenicity study required to qualify a new MR or requalify 

the current MR

3) Scenario 3

• Modern product MRs stored frozen

• Passes FDFT regimen

• Monitoring via RSM assay and serial release data, submitted every 2 1⁄2 years

• 15-year initial dating during which RSM plan must be approved and RSM 

assays validated
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• Reference qualification/requalification via host animal immunogenicity study 

if time zero testing not conducted within 2 years of original MR qualification

4) Scenario 4

• Modern product MRs stored frozen

• Fails FDFT regimen

• Monitoring via RSM assays and serial release data

• Dating assigned in 5-year increments based on process toward approval of 

RSM plan and RSM assays validation – Maximum 15 years

• Reference qualification/requalification via host animal immunogenicity study 

required

5) Scenario 5

• Modern product frozen MRs

• Monitoring via RSM assays and serial release data, submitted every 2 1⁄2 years

• Required when FDFT regimen fails or can be optionally chosen for the benefits 

of dating and reference qualification in vitro

• RSM plan approved, RSM assays validated, time zero testing conducted within 

2 years of MR qualification in host animals

• Indefinite dating possible

6) Scenario 6

• Modern product MR not frozen, product storage

• Monitoring via RSM assays and serial release data, submitted every 2 1⁄2 years

• Product dating prior to approved RSM plan and validating RSM assays

• Reference qualification/requalification host animal immunogenicity study

required prior to monitoring using RSM assays from time zero

7) Scenario 7

• Modern product MR not frozen, product storage

• Monitoring via RSM assays and serial release data, submitted every 2 1⁄2 years

• Approved RSM plan and validating RSM assays, testing initiated at time zero

• Reference qualification in vitro
U.S. DEPARTMENT OF AGRICULTURE 

Marketing and Regulatory Programs

Animal and Plant Health Inspection Service

Veterinary Services 

VS Memorandum (VSM) 800.211 

__________________________________________________________________ 

15 | Page

7. Implementation/Applicability 

Updated policy in this memorandum is effective immediately.